Clusterin Antibody

What is Clusterin and why are antibodies against it important for research?

Clusterin is a heterodimeric glycoprotein (75-80 kDa) found in most biological fluids and tissues throughout the body. It exists in multiple isoforms and plays roles in cell death regulation, tumor progression, and neurodegenerative disorders . The protein consists of alpha and beta chains derived from a common precursor.

Antibodies against Clusterin are important research tools because:

• They enable detection and quantification of Clusterin in various samples and experimental conditions

• They help investigate Clusterin's involvement in pathological processes including cancer, Alzheimer's disease, and inflammatory conditions

• They allow researchers to distinguish between different Clusterin isoforms and their specific functions

• They provide insights into Clusterin's subcellular localization and tissue distribution

Different antibodies recognize specific epitopes within the Clusterin protein, including the alpha chain (Ser228-Glu449) and beta chain (Asp23-Arg227), enabling selective targeting of different functional domains .

What are the key differences between monoclonal and polyclonal Clusterin antibodies for research applications?

The choice between monoclonal and polyclonal Clusterin antibodies significantly impacts experimental outcomes:

What molecular weight bands should I expect when using Clusterin antibodies in Western blot?

When performing Western blot analysis with Clusterin antibodies, researchers should anticipate multiple bands depending on the sample type and reducing conditions:

• Clusterin precursor: 60-65 kDa band

• Clusterin alpha chain: approximately 36 kDa

• Clusterin beta chain: approximately 36-37 kDa

Published scientific data shows that under reducing conditions, antibodies like MAB2937 detect both the 60-65 kDa precursor and the 36 kDa alpha chain in human liver tissue and serum samples . Similarly, AF2937 detects both the precursor and the cleaved forms in Western blots .

The apparent molecular weight may vary slightly depending on:

• Post-translational modifications (particularly glycosylation)

• Sample preparation method

• Reducing vs. non-reducing conditions

• Tissue or cell type (due to tissue-specific modifications)

• Antibody specificity for particular domains

When troubleshooting unexpected band patterns, consider that some antibodies may preferentially recognize either the mature cleaved forms or the full-length precursor depending on their epitope recognition sites .

How should I optimize immunohistochemistry protocols when using Clusterin antibodies?

Optimizing immunohistochemistry (IHC) protocols for Clusterin detection requires careful consideration of several parameters:

Tissue Preparation and Fixation:

• Both paraffin-embedded and frozen sections can be used with appropriate antibodies

• For paraffin sections, standard formalin fixation works well with many Clusterin antibodies

• For frozen sections, immersion fixation shows good results with antibodies like MAB2937

Antibody Selection and Dilution:

• Start with manufacturer-recommended concentrations (typically 1-5 μg/mL)

• For MAB29372, 1 μg/mL has shown specific staining in human liver sections

• For MAB2937, 5 μg/mL has shown specific staining in mouse spleen and human prostate cancer tissue

• Always perform a dilution series (0.5-10 μg/mL) to optimize signal-to-noise ratio

Detection System:

• HRP-polymer detection systems (e.g., VisUCyte™ HRP Polymer) show excellent results with DAB visualization

• For fluorescent detection, select secondary antibodies compatible with your primary antibody's host species

Antigen Retrieval:

• Heat-induced epitope retrieval using citrate buffer (pH 6.0) is generally effective

• For challenging tissues, try alternative buffers (Tris-EDTA, pH 9.0)

Protocol Optimization Steps:

• Test multiple antibody concentrations

• Vary antigen retrieval methods and times

• Adjust primary antibody incubation time (1 hour at RT or overnight at 4°C)

• Include proper positive controls (liver, prostate tissue) and negative controls

• For double-labeling experiments, test for cross-reactivity between detection systems

Clusterin typically shows cytoplasmic and/or membranous staining patterns, with specific localization varying by tissue type .

What are the optimal conditions for detecting Clusterin in Western blot experiments?

Successful Western blot detection of Clusterin requires attention to several technical aspects:

Sample Preparation:

• Human liver tissue and serum samples serve as excellent positive controls

• Use RIPA or immunoblot buffer (Group 1) for efficient protein extraction

• Include protease inhibitors to prevent degradation

Gel Electrophoresis and Transfer:

• Use 10-12% SDS-PAGE gels for optimal resolution of Clusterin bands

• PVDF membranes typically yield better results than nitrocellulose for Clusterin detection

• Transfer conditions: 100V for 1 hour or 30V overnight for larger proteins

Blocking and Antibody Incubation:

• 5% non-fat dry milk or 5% BSA in TBST works well for blocking

• Recommended antibody dilutions:

  • MAB2937: 0.5 μg/mL for Western blots

  • AF2937: 1 μg/mL for Western blots

  • Rabbit polyclonal (12289-1-AP): 1/1000 dilution

• Incubate primary antibody overnight at 4°C for optimal sensitivity

Detection Strategy:

• HRP-conjugated secondary antibodies work well with most Clusterin primaries

• For mouse monoclonal antibodies like MAB2937, use anti-mouse IgG secondary (e.g., HAF018)

• For goat polyclonal antibodies like AF2937, use anti-goat IgG secondary (e.g., HAF017)

Expected Results:

• Under reducing conditions, expect bands at 60-65 kDa (precursor) and ~36 kDa (alpha/beta chains)

• A549 cells show a predominant band at approximately 65 kDa with rabbit anti-clusterin

• Comparison of expected bands across sample types:

How can I develop a reliable ELISA for Clusterin quantification?

Developing a robust ELISA for Clusterin quantification requires careful consideration of several factors:

Antibody Pair Selection:

• Use a capture-detection antibody pair that recognizes different epitopes

• Human Clusterin monoclonal antibodies (e.g., clone #350270) have been validated for sandwich immunoassays

• Consider using a monoclonal capture antibody with a polyclonal detection antibody for improved sensitivity

Assay Format Options:

• Direct ELISA: Immobilize sample, detect with labeled anti-Clusterin antibody

  • Simpler but typically less sensitive than sandwich format

  • Several antibodies including MAB2937 have been validated for direct ELISA

• Sandwich ELISA: Capture antibody → sample → detection antibody → substrate

  • Higher specificity and sensitivity

  • Reduced matrix effects from complex samples

Protocol Optimization:

• Coating concentration: Test 1-10 μg/mL of capture antibody

• Blocking: 1-5% BSA or commercial blocking buffers

• Sample dilution: Create a dilution series to ensure readings fall within standard curve

• Detection antibody concentration: Follow manufacturer recommendations, typically 0.1-1 μg/mL

• Standard curve: Use recombinant human Clusterin with 7-8 point standard curve (2-fold dilutions)

Validation Checks:

• Analytical specificity: Confirm minimal cross-reactivity with related proteins like Clusterin-like 1

• Precision: Intra-assay CV <10%, inter-assay CV <15%

• Recovery testing: Spike known amounts of Clusterin into samples

• Parallelism: Ensure diluted samples maintain linearity

Sample Considerations:

• Serum/plasma samples typically contain high levels of Clusterin

• Cell culture supernatants may require concentration

• Tissue homogenates should be prepared in appropriate buffers with protease inhibitors

For highest sensitivity, consider biotinylated detection antibodies with streptavidin-HRP for signal amplification.

What are the common sources of false positive and false negative results when using Clusterin antibodies?

Understanding potential sources of error is crucial for accurate interpretation of Clusterin antibody-based experiments:

Common Causes of False Positive Results:

Common Causes of False Negative Results:

Interpretive Challenges:

• Multiple Clusterin isoforms can create complex banding patterns

• Tissue-specific expression and modification can alter expected results

• Disease states can significantly alter Clusterin expression patterns

For the most reliable results, include both positive controls (e.g., human liver tissue, serum) and negative controls in each experiment .

How can I distinguish between different Clusterin isoforms using antibodies?

Distinguishing between Clusterin isoforms requires careful selection of antibodies and experimental approaches:

Major Clusterin Isoforms:

• Secreted Clusterin (sCLU): The predominant form (~80 kDa precursor, processed into ~40 kDa α and β chains)

• Nuclear Clusterin (nCLU): Alternatively spliced variant (~55 kDa)

• Cytoplasmic Clusterin (cCLU): Intracellular variant

Antibody Selection Strategies:

• Epitope-Specific Antibodies:

  • Choose antibodies targeting unique regions of specific isoforms

  • Antibodies recognizing Asp23-Arg227 (beta chain) and Ser228-Glu449 (alpha chain) regions can help identify specific forms

• Combining Multiple Antibodies:

  • Use a panel of antibodies targeting different epitopes

  • Compare staining/detection patterns across experiments

Experimental Approaches:

• Western Blot Analysis:

  • Use appropriate gel percentage (10-12%) for optimal separation

  • Compare molecular weights: sCLU (60-65 kDa precursor, 36 kDa chains), nCLU (~55 kDa)

  • Include subcellular fractionation to separate nuclear and cytoplasmic compartments

• Immunofluorescence Microscopy:

  • Perform co-localization studies with compartment markers

  • sCLU: Co-localizes with secretory pathway markers (Golgi, ER)

  • nCLU: Shows nuclear localization

  • cCLU: Diffuse cytoplasmic pattern

• Flow Cytometry:

  • Distinguish between cell surface (secreted) and intracellular forms

  • Requires membrane permeabilization to detect internal forms

Validation Approaches:

• Use recombinant isoform standards as controls

• Consider siRNA knockdown experiments targeting specific isoform transcripts

• Verify with mass spectrometry for definitive isoform identification

The secreted form of Clusterin has been most extensively studied, particularly in cancer research, where it has been found to promote epithelial-mesenchymal transition and chemoresistance .

What controls should I include when working with Clusterin antibodies?

Robust experimental design requires appropriate controls to validate findings with Clusterin antibodies:

Essential Positive Controls:

• Tissue/Cell Controls:

  • Human liver tissue: Consistently shows strong Clusterin expression

  • Human prostate cancer tissue: Shows cytoplasmic Clusterin staining

  • Mouse spleen: Suitable for cross-reactive antibodies

  • A549 cells: Show a ~65 kDa Clusterin band in Western blots

• Sample-Type Controls:

  • Human serum: Contains high levels of secreted Clusterin

  • Include known Clusterin-expressing samples relevant to your research context

Critical Negative Controls:

• Antibody Validation Controls:

  • Isotype control antibodies (same species, isotype as primary)

  • Secondary antibody-only controls

  • Peptide competition assays to demonstrate specificity

• Biological Negative Controls:

  • Tissues/cells known to express minimal Clusterin

  • siRNA or CRISPR knockdown samples (when feasible)

  • Samples from CLU-knockout models (if available)

Specificity Validation Controls:

• Cross-Reactivity Assessment:

  • Test against related proteins (e.g., Clusterin-like 1, mouse Clusterin)

  • Confirm antibody specificity across species if working with non-human models

• Application-Specific Controls:

  • For IHC: Include no-primary antibody sections

  • For WB: Include molecular weight markers

  • For IP: Include IgG control immunoprecipitation

  • For ICC/IF: Include peptide blocking controls

Data Interpretation Controls:

• Technical Replicates:

  • Run at least 3 technical replicates for quantitative applications

  • Ensure consistent results across replicates

• Reference Standards:

  • Include recombinant Clusterin standards at known concentrations

  • Consider using different antibody clones to confirm findings

Properly documented controls significantly enhance the reliability and reproducibility of Clusterin antibody-based research and should be reported in all publications.

How can Clusterin antibodies be used to investigate epithelial-mesenchymal transition (EMT) in cancer?

Clusterin plays a significant role in promoting epithelial-mesenchymal transition (EMT), a critical process in cancer progression and metastasis. Antibody-based approaches offer valuable insights into this process:

Mechanistic Role of Clusterin in EMT:

• Secreted Clusterin (sCLU) stimulates EMT in cancer cells

• This leads to increased tumor invasion, metastasis, and chemoresistance

• The EMT-inducing region in sCLU is a key therapeutic target

Antibody-Based Research Strategies:

• Therapeutic Antibody Development:

  • AB-16B5, a humanized IgG2 mAb, specifically targets the EMT-inducing region of sCLU

  • This antibody has shown inhibition of EMT in pre-clinical models and synergy with chemotherapeutic agents

  • Phase I clinical trials have demonstrated its safety profile in patients with advanced solid tumors

• Monitoring EMT Status:

  • Use Clusterin antibodies in conjunction with EMT markers (E-cadherin, vimentin, N-cadherin)

  • Quantify changes in Clusterin expression during EMT progression

  • Perform co-localization studies to track Clusterin redistribution during EMT

• Experimental Approaches:

  • IHC analysis of tumor biopsies before and after treatment

  • Western blot quantification of Clusterin and EMT markers

  • Immunofluorescence to track cellular localization changes

Clinical Research Applications:

• Pre-treatment and post-treatment tumor biopsies can be analyzed using Clusterin antibodies to evaluate EMT status

• This approach has been implemented in clinical trials to investigate the efficacy of anti-Clusterin therapies

• Changes in Clusterin expression patterns may serve as biomarkers for treatment response

Experimental Design Considerations:

• Include multiple EMT markers alongside Clusterin

• Consider time-course experiments to track EMT progression

• Use cell line models with well-characterized EMT phenotypes

• Combine with functional assays (migration, invasion) to correlate expression with behavior

The development of AB-16B5 represents a significant advance in translating basic Clusterin research into potential therapeutic applications, with phase I trials showing manageable safety profiles and establishing groundwork for further clinical investigation .

What role do Clusterin antibodies play in neurodegenerative disease research?

Clusterin antibodies are invaluable tools in investigating the complex role of this protein in neurodegenerative disorders, particularly Alzheimer's disease:

Clusterin in Neurodegenerative Pathology:

• Clusterin co-localizes with amyloid plaques in Alzheimer's disease

• It interacts with alpha-synuclein in Parkinson's disease

• Genetic studies have identified CLU as a risk factor for Alzheimer's disease

Antibody Applications in Neurodegeneration Research:

• Neuropathological Analysis:

  • Clusterin antibodies like AF2937 have been used to detect Clusterin in Alzheimer's disease brain tissue

  • These studies reveal Clusterin's association with pathological structures

• Protein Interaction Studies:

  • Antibodies help investigate Clusterin's interaction with:

    • Amyloid-beta peptides in Alzheimer's disease

    • Alpha-synuclein in Parkinson's disease

    • Tau protein in tauopathies

• Biochemical Characterization:

  • Western blot analysis of brain tissue reveals Clusterin expression patterns

  • Immunoprecipitation with anti-Clusterin antibodies enables analysis of protein complexes

  • Changes in Clusterin isoform distribution can be tracked during disease progression

Experimental Evidence:

• Clusterin has been shown to interact with alpha-synuclein preformed fibrils (pffs) in primary astrocytes

• Western blot analysis using Clusterin antibodies demonstrated increased Clusterin levels in culture medium from astrocytes treated with alpha-synuclein pffs compared to monomeric alpha-synuclein

• Quantification normalized to GAPDH showed significant differences (p=.0028) between treatment conditions

Research Methodologies:

• Brain tissue immunohistochemistry with antibodies like AF2937

• Co-immunoprecipitation to isolate Clusterin-protein complexes

• Double-labeling immunofluorescence to examine co-localization

• Western blot analysis of tissue homogenates from various brain regions

This research area highlights the value of using multiple antibodies targeting different Clusterin epitopes to comprehensively investigate its role in neurodegenerative mechanisms.

How can researchers use Clusterin antibodies to investigate cancer therapeutics and biomarkers?

Clusterin antibodies are powerful tools for cancer research, offering insights into both therapeutic targeting and biomarker development:

Clusterin as a Cancer Target and Biomarker:

• Clusterin promotes cancer cell survival and treatment resistance

• Its expression is upregulated in various cancers, including prostate, breast, and lung cancer

• Clusterin inhibition can sensitize cancer cells to therapy

Therapeutic Applications:

• Development of Anti-Clusterin Therapeutics:

  • AB-16B5, a humanized IgG2 monoclonal antibody, specifically targets the EMT-inducing region of sCLU

  • Phase I clinical trials demonstrated its safety profile in patients with advanced solid tumors

  • The trial enrolled 15 patients with various carcinomas, melanoma, and sarcoma

  • Patients received between 1 and 53 weekly doses (median: 9 doses)

  • Most adverse events were Grade 1 or 2, with only two Grade 3 events (infusion-related reaction and rash) related to AB-16B5

• Combination Therapy Research:

  • Clusterin antibodies help investigate synergistic effects with chemotherapeutic agents

  • Pre-clinical models showed AB-16B5 displays synergy with chemotherapy

  • Monitoring Clusterin expression can help identify optimal treatment combinations

Biomarker Applications:

• Expression Analysis in Clinical Samples:

  • IHC with antibodies like MAB2937 can detect Clusterin in cancer tissues

  • This antibody has been validated for human prostate cancer tissue analysis

  • Expression patterns correlate with clinical parameters and outcomes

• Circulating Clusterin Detection:

  • Antibodies enable quantification of serum/plasma Clusterin levels

  • Sandwich immunoassays using antibodies like clone #350270 provide sensitive detection

  • Changes in circulating Clusterin may indicate disease progression or treatment response

Methodological Approaches:

• Tissue Analysis:

  • IHC staining of tumor tissue microarrays (TMAs)

  • Scoring systems based on staining intensity and distribution

  • Correlation with clinicopathological parameters

• Liquid Biopsy Development:

  • ELISA-based detection in serum/plasma

  • Multiplex assays combining Clusterin with other cancer biomarkers

  • Longitudinal monitoring during treatment

• Functional Studies:

  • Antibody-mediated neutralization in cell culture models

  • Xenograft studies with anti-Clusterin antibodies

  • Combination studies with standard-of-care treatments

The clinical development of AB-16B5 represents a significant translation of basic Clusterin research to therapeutic applications, demonstrating the value of targeting this protein in cancer treatment strategies .

What are the emerging applications of Clusterin antibodies in inflammation and immunology research?

Clusterin's roles in inflammation and immune regulation present emerging opportunities for antibody-based research:

Clusterin in Inflammatory Processes:

• Functions as an extracellular chaperone in response to stress

• Modulates complement activation and immune complex clearance

• Influences cytokine production and inflammatory signaling

Antibody Applications in Inflammation Research:

• Tissue Distribution Studies:

  • Clusterin antibodies like MAB2937 and MAB29372 detect Clusterin in immune tissues such as spleen

  • IHC reveals specific localization patterns in immune cell subpopulations

  • Different antibody clones may reveal distinct aspects of Clusterin biology in immune contexts

• Regulation of Complement System:

  • Antibodies help investigate Clusterin's interaction with complement components

  • Co-immunoprecipitation experiments reveal protein-protein interactions

  • Functional assays combined with antibody detection track complement regulatory activity

• Inflammatory Disease Models:

  • Monitor Clusterin expression changes during disease progression

  • Correlate with inflammatory markers and clinical parameters

  • Evaluate potential as therapeutic target in inflammatory conditions

Methodological Approaches:

• Multi-parameter Flow Cytometry:

  • Combine Clusterin antibodies with immune cell markers

  • Analyze expression in different leukocyte populations

  • Track changes during inflammatory activation

• Cytokine-Clusterin Interactions:

  • Use antibodies to investigate how inflammatory cytokines regulate Clusterin

  • Examine Clusterin's effects on cytokine production and signaling

  • Develop co-culture systems with antibody-based detection

• In vivo Imaging Applications:

  • Develop labeled Clusterin antibodies for in vivo tracking

  • Monitor inflammation sites and Clusterin distribution

  • Evaluate therapeutic responses in real-time

Research Considerations:

• Different inflammatory conditions may alter Clusterin glycosylation and processing

• Consider using multiple antibody clones recognizing different epitopes

• Integrate with systems biology approaches to understand network effects

This emerging field highlights the versatility of Clusterin antibodies beyond traditional cancer and neurodegeneration research, opening new avenues for understanding and potentially treating inflammatory disorders.

What are the potential applications of novel Clusterin antibody formats in research and therapeutics?

The evolution of antibody engineering technologies presents exciting opportunities for developing next-generation Clusterin antibodies:

Emerging Antibody Formats:

• Bispecific Antibodies:

  • Simultaneous targeting of Clusterin and tumor-associated antigens

  • Enhanced recruitment of immune effector cells to Clusterin-expressing tumors

  • Improved tissue penetration and therapeutic efficacy

• Antibody-Drug Conjugates (ADCs):

  • Clusterin antibodies as delivery vehicles for cytotoxic payloads

  • Targeted delivery to Clusterin-overexpressing cancer cells

  • Potential for reduced off-target effects in cancer therapy

• Intrabodies and Nanobodies:

  • Engineered for intracellular targeting of Clusterin

  • Potential to modulate intracellular Clusterin functions

  • Enhanced tissue penetration and reduced immunogenicity

Research Applications:

• Advanced Imaging:

  • Site-specific fluorophore-conjugated antibodies for super-resolution microscopy

  • Multicolor imaging of Clusterin isoforms and interacting partners

  • Intravital imaging of Clusterin dynamics in disease models

• Proximity-Based Methods:

  • Antibody-based proximity ligation assays for protein interactions

  • BioID or APEX2 fusion antibodies for proximity labeling

  • Split-reporter systems coupled to Clusterin-binding fragments

Therapeutic Development Considerations:

• Humanization Strategies:

  • The AB-16B5 humanized IgG2 mAb provides proof-of-concept for therapeutic targeting

  • Future antibodies may benefit from enhanced humanization techniques

  • Fc engineering for optimized effector functions or extended half-life

• Combination Approaches:

  • Pre-clinical models showed AB-16B5 displays synergy with chemotherapy

  • Future research may explore combinations with immunotherapy

  • Dual targeting of multiple cancer survival pathways

Technical Challenges:

• Maintaining epitope specificity during antibody engineering

• Ensuring adequate tissue penetration for solid tumor applications

• Addressing potential immunogenicity of novel formats

The clinical development of AB-16B5 has established important groundwork for future therapeutic Clusterin antibodies, demonstrating acceptable safety profiles in phase I trials with advanced cancer patients .

How might advances in antibody technology enhance Clusterin research in single-cell and spatial transcriptomics?

The integration of antibody technologies with emerging single-cell and spatial analysis methods presents transformative opportunities for Clusterin research:

Single-Cell Analysis Applications:

• CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing):

  • Oligonucleotide-tagged Clusterin antibodies enable simultaneous protein and mRNA analysis

  • Correlation of Clusterin protein expression with transcriptome-wide changes

  • Identification of cell populations with discordant mRNA/protein expression

• CyTOF (Mass Cytometry):

  • Metal-conjugated Clusterin antibodies for high-parameter single-cell analysis

  • Simultaneous detection of Clusterin with dozens of other proteins

  • Deep phenotyping of Clusterin-expressing cells in heterogeneous tissues

Spatial Analysis Methods:

• Multiplexed Immunofluorescence:

  • Cyclic immunofluorescence with Clusterin antibodies

  • Co-localization analysis with multiple markers in situ

  • Preservation of spatial context critical for understanding Clusterin function

• In Situ Sequencing with Antibody Detection:

  • Combining antibody staining with spatial transcriptomics

  • Mapping Clusterin protein distribution relative to its mRNA

  • Understanding post-transcriptional regulation in tissue context

Methodological Considerations:

• Antibody Validation Requirements:

  • Additional specificity validation for single-cell applications

  • Optimization of antibody concentrations for multiplexed detection

  • Cross-platform validation to ensure consistent results

• Technical Challenges:

  • Signal amplification for low-abundance detection

  • Antibody panel design to minimize spectral overlap

  • Data integration across protein and transcript measurements

Potential Research Applications:

• Tumor Heterogeneity Analysis:

  • Mapping Clusterin expression across distinct tumor microenvironments

  • Identifying therapy-resistant niches with altered Clusterin expression

  • Spatial relationship between Clusterin-expressing cells and immune infiltrates

• Neurodegenerative Disease Investigation:

  • Spatial distribution of Clusterin relative to pathological structures

  • Cell type-specific expression patterns in affected brain regions

  • Microenvironmental factors influencing Clusterin expression

These emerging technologies will significantly enhance our understanding of Clusterin's complex roles in health and disease by providing unprecedented resolution at both single-cell and spatial levels.